R.K.Singh Plant Breeding Course, April 06 Breeding for Salt Tolerance in Rice R.K. Singh PBGB, IRRI.

R.K.SinghPlant Breeding Course, April 06

Breeding for Salt Tolerance in Rice

R.K. SinghPBGB, IRRI


Extent of the problem and management options

Reason of Limited Success Plant adaptation – salt

tolerant mechanisms Morphological symptoms Basic concepts (genotype vs.

phenotype and heritability) Genetic Studies Screening techniques Breeding strategy Physiological mechanisms Molecular mapping Varietal development NRM approaches

Outlines of the Lecture


EXTENT OF SALT-AFFECTED SOILS

World’s Total area12.78 b ha

340 x 106 ha (Ponamperuma, 1984)954 x 106 ha ( Massoud, 1974)10% area ~ 1.2 b ha (Tanji, 1991)

Sodic

Saline

FAO Database397 x 106 ha (3.1%) – Saline soils434 x 106 ha (3.4 %)– Sodic Soils

Asia, Pacific and Asia, Pacific and Australia (M ha)Australia (M ha)

195 249

Source : FAO databaseTotal : 444 M ha


Salt-affected type

Electrical conductivity

ECe

(dSm-1)#

Exchangeable Sodium

Percentage ESP (%)

Sodium Absorption

Ratio SAR

pHs

Saline > 4 < 15 < 13 < 8.8 Sodic < 4 > 15 > 13 8.5-10.5 Saline - sodic > 4 > 15 Variable > 8.5

# at 25C

ESP = Exchangable Sodium x 100 Cation Exchange Capacity

SAR

Na

Ca Mg

2 2

2

What are the salt-affected soils ?


How to Manage the Salt-affected Areas ?

1. Environment modifying approach : Change the environment for the normal growth of plants

2. Crop based approach : Select or develop crop variety which can withstand the salt stress

Do we need ST

cultivars ?

Rice has enormous variability


Management of the Salt-affected Soils

3. Hybrid Approach

It is the combination of environment modifying and plant based approach.

Advantages:• More viable• Highly productive• Low resource cost

Local variety without gypsum

Salt tolerant rice variety, CSR13, with 25% Gypsum


Reasons of Limited Success

Salt stress seldom happen in isolation

Harsh, highly variable environment, large G/E

Lack of efficient / precise screening procedure

Lack of mechanistic understanding

Low priority and less number of researchers involved


Salt Stresses and Associated Complexities

SALT STRESSES

Acid

SO4

Peat

SALINE

ALKALINE

INLAND SALINE

(P, Zn)

(P, Zn)

(P, Zn)

(P, Zn)

(Fe)

Fe, Altox

Fe, H2Stox

Al, OrganicAcids tox

(P & Zn)

RAINFED

Sub-merged

Deep-water

Drought

Irrigated

GraIn

Quality

(Source: Glenn B. Gregorio)


Breeding for

Salt tolerance + High productivity

• Na+ Exclusion• Tissue tolerance• K+ uptake• Partitioning etc.

All are quantitative trait

Quantitative trait

Single trait


1. Restricting the entry of toxic ions at root level - Exclusion

2. Transporting the toxic ions to stem, leaf sheath or older leaves – plant level compartmentation

4. Sequestration of the toxic ions to vacuole or cell wall – cell level compartmentation

3. Excretion of salt through salt glands, salt-hairs or bladders – in most halophytes

Predominant salt-tolerance mechanisms operating in plant

Na+ Cl-


Physiology: traits associated with salinity tolerance

Regulation of uptake

Compartmentation In old tissue

Upregualtion of antioxidants

Vigorous growth

Responsive stomata

[Na+]

OsmoprotectantsAOSS

K+

AtNHX1H+Na+

Vacuolar Na+/H+

SOS1Na+ H+

Plasma Na+/H+

AVP1H+

PPiase

Compartmentation within tissue(tissue tolerance)

Protective metabolites

Polyamines, dehydrins, glyoxalates

Earliness

Source : A.M. Ismail


Morphological Symptoms White leaf tip followed by tip burning (salinity) Leaf browning & death (sodicity) Stunted plant growth Low tillering Spikelet sterility Low harvest index Less florets per panicle Less 1000 grain weight Low grain yield Change in flowering duration Leaf rolling White leaf blotches Poor root growth Patchy growth in field

Manifestation of Salt Stress


First symptom“Leaf tip burning”

“Leaf tip burning extends toward base through

Lamina”

“Ultimate death of leaf – always from oldest to

youngest”

Salinity symptoms at the vegetative stage


Effect of salinity at Reproductive stage – Spikelet Sterility


Effect of salinity at reproductive stage – papery sterile spikelets


Physiological & Biochemical High Na+ transport to shoot Preferential accumulation of Na in older leaves High Cl- uptake Lower K+ uptake Lower fresh and dry weight of shoot and roots Low P and Zn uptake Increase of non-toxic organic compatible solutes Increase in Polyamine levels

Manifestation of Salt Stress

Screening parameters ?


Which is the most reliable stage for screening ?

Association between

Correlation Coeff.

Glasshouse studies

Field studies

Veg. stage tolerance vs. Grain yield

Rep. stage tolerance vs. Grain yield

Veg. stage vs. Rep. stage tolerance

- 0.58ns

- 0.97**

0.59ns

- 0.022ns

- 0.82**

0.34ns

Vegetative vs. Reproductive stage salt tolerance


Chaffy panicles

Papery florets

IR66946-3R-178-1-1


Number of gene(s) responsible for a trait (n) / Genotypic classes

1 2 5 10

F2

F3

F4

F5

F6

1:2:1

3:2:3

7:2:7

15:2:15

31:2:31

1:2:1:2:4:2:1:2:1

3:2:3:6:4:6:3:2:3

7:2:7:14:4:14:7:2:7

15:2:15:30:4:30:15:2:15

31:2:31:62:4:62:31:2:31

243

classes

59,049

classes

P* 1/4 1/16 1/ 1,024 1/ 1,084,576

*: P is the probability of getting the desired homozygote at all the loci in smallest perfect population in F2 (1/4n)

Trait A = 5 loci -- Desired recombinant – 1/1,024Trait B = 10 loci -- 1/1,084,576Prob. of getting both desired one in one background = 1/1,024 x 1/1,084,576 = 1/ 1,110,605,824 (> 1b)

Probability of getting the desirable genotype

Why Recurrent selection – mating of the selected individuals ?


Precision vs. Resources

Precision

l

r

y

Resources

No. of

low

more

Very High

Since the salinity is highly variable in soil due to the dynamic state of soluble salts hence one should go for more blocks at different locations over the years (judiciously compromising the resources) for the precise estimates


Based on reproductive stage tolerance

Bas. 370 / CSR10

Bas. 370 / CSR11 Pak. Bas. / CSR10

Controlled by numerous minor genes as revealed by the normal distribution curve with few major genes (skewness)

SALINITY

Substituted

Genetics of Salt ToleranceInheritance Pattern


Inheritance for sodicity tolerance

Similar results (based on the same crosses

Genetics of Salt Tolerance

(a) Bas.370 x CSR10

0100

200300

400500

600700

1 3 5 7 9

n=1689

Freq

uenc

y

(b) Bas.370 x CSR 11

0

20

40

60

80

100

120

140

1 3 5 7 9

n=306

Freq

uenc

y

(c) Pak. Bas x CSR10

0

50

100

150

200

250

300

350

1 3 5 7 9

Score

Freq

uenc

y

Frequency

Trend

n=443F1

P1XP2 F1

XF1 X

P1


Genetic Estimates of Parameters Genetic Parameters

Salinity tolerance

score (R) K+ Na+/K+

Variation due to additive effects (D) Variation due to dominance effects (H1) Degree of Dominance ((H1/D)½ ) Heritability in narrow sense (h2

ns)

10.760*1.080

7.532*2.916

0.837

0.65

0.056*0.009

0.067*0.024

1.091

0.50

0.099*0.017

0.129*0.044

1.139

0.41

SV df Mean Square

GCA

SCA

Error

5

15

40

15.36*

3.71*

0.22

0.238*

0.066*

0.012

0.100*

0.033*

0.006

Genetics of Salt toleranceGene Action (based on 6x6 diallel)


Seedling stage tolerance:In 20 days can classify the tolerance level

Screening Techniques Standardised


Screening technique standardised

KR

1-

24

Tol

eran

t ch

eck

IR 6

6946

-3R

-178

-1-1

IR 2

9 (s

ensi

tive

che

ck)

Mu

skan

41

Ch

eriv

irup

pu

IR 6

6946

-3R

-178

-1- 1

IR 2

9NB: Instead of Pokkali, now IR 66946-3R-178-1-1 is being used as tolerant check which is derived from IR29 / Pokkali cross. It is semi-tall, photoinsensitive and highly salt tolerant


Normal

Saline

1 2 3

1 2 3

FL478 / IR29 FL478 / IR29 FL478 / IR29

FL478 / IR29 FL478 / IR29 FL478 / IR29

Performance of 1 mo-old FL478 (tolerant line) and IR29 (susceptible variety) rice seedlings under normal and saline (14d EC12 then 14d EC18) conditions using SNAP and nutrient solutions: (1) 100% SNAP solution in tap water, (2) 75% SNAP solution in tap water, and (3) nutrient solution in distilled water.

(Source: Dante Adorada)


Comparison between 28-day old rice seedling grown for 21 days in SNAP solution (Simple Nutrient Addition Program) with (a) 100% nitrate and (b) 90% nitrate & 10% ammonium in

their composition.

(Source: Dante Adorada)


Phenotyping for the Adult Plant Salinity Tolerance

Microplots with controlled salinity and sodicity

Sodic Soil EnvironmentSaline Soil Environment

(Rain shelter)

Automatic Circulatory Solution Culture System


Na is the most notorious element causing salt related problems in plants

Its higher uptake hinders the metabolic activities in plants

Plants try to resist this element using various physiological mechanisms

• Na+ exclusion, • Tissue Tolerance • Higher K+ uptake to counter Na • Compartmention (Preferential accumulation of Na+ in stem,

leaf sheath, older leaves etc.) • Early vigour • …… Many more

Salinity Tolerance in Rice


Breeding Strategy

Identification of the genotypes based on the inherent physiological mechanism (Na exclusion, K uptake, Tissue tolerance and high initial vigor etc.) responsible for salinity tolerance

Inter-mating of the genotypes with high degree of expression of the contrasting salinity tolerance mechanism

Identifying / screening of the recombinants for pooling/ pyramiding of the mechanisms


Identify the donors for predominant physiological mechanisms responsible for salt tolerance

• Na+ exclusion, • Tissue Tolerance • K+ uptake, • Preferential accumulation of Na+ in stem, leaf sheath, older

leaves etc. • Early vigour

However, none of the rice variety posses all the possible positive mechanism conferring salinity tolerance.

Breeding Strategy


Grouping of the rice varieties on the basis of Na accumulation per day

Bas.370, CSR10, CSR19

M I-48, Bas.385,CSR18, PR108

Low< 0.1 m m ol/g

CSR11, IR36, HBC19, CSR20, ADT36HKR128, CSR1, Jaya, CSR13, Ach h i

Su kh vel, IR42, IR24, Majh era7, Man g laSLR51214, P rasad , Van d n a, Salivah n a

M edium0.1 - 0 .499 m m ol/g

SR26B, CSR21, IR4630, Pokkali, T-23G R11, Pan vel-2, In d rasan , IR 58, RP144Hath wan , Carp s C lark, Swarn d h an , RaviUd aya, T-21, Majh era-3, Barkat, MK47-22

High> 0.5 m m ol

Na accum ulation per day(m m ol/g dw t)


CSR21, IR4630, Hath w an , Sw arn d h a nIn d rasan , Ach h i, M u skan , Ud aya

RP144, VKL -3 9, Carp s C lark

High> 0.4 mm ol/g

CSR11, IR 36, CSR 20, Panv el-23, BC 1Ravi, Sa livahan a, Hasa n Sarai, Barka tPR106, IR58, IR24 , M ajhera-3, ADT 3 6

SLR51214, IR 42, M K47-22 , T -23, M angla

M edium0.2 - 0.4 mm ol/g

SR26B , HBC 19, CSR1, Pokkali, T -2 3G R11, PR10 8, CSR10, CS R18, CSR1 9Jaya, HKR128, M I-48, Bas.370, Bas.38 5M ajhera-7, P rasad, Vandn a, V ikram ary a

Low< 0.2 m m ol

K accumulation per day(m m ol/g dw t)

Grouping of the rice varieties on the basis of K accumulation per day


Na/K ratio in different rice varieties

0.01.02.03.04.05.06.07.08.09.0

10.0

PR 1

08

P-20

3

CSR

19

HB-

101B

MTU

AR

203

8

Has

an S

arai

CSR

1

VK

L-39

CSR

18

CSR

20

CSR

10

IR 4

2

Deh

radu

ni IR36

CSR

11

Rat

nagi

ri

Bark

at

Hat

hwan

HBC

19

Panv

el-3

3

Maj

hera

-7

Indr

asan

9310

9

Pusa

-150

Uda

ya

Kal

ing-

2

P-11

76-9

1-1-

2-2

SR 2

6B

POK

KA

LI

Pusa

129

8-1

Vik

ram

arya

IR61

311-

3B-8

Sarb

ati

Pak-

803

CSR

8

IR 2

153

JAY

A

HK

R 1

28

Varieties

Na/

K r

atio


Na and K accumulation in rice varieties on per day basis

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Varieties

Na &

K (m

mol

/g/d

ay)

0

2

4

6

8

10

12

14

16

18

Trea

tmen

t day

s

Na Accumulation K Accumulation Days of treatment


Rice variety AGood excluder +poor tissue tolerance

Rice variety BPoor control at root level + High tissue tolerance

Dustbin

GarbageNa+

Rice variety C

Good excluder+

High tissue tolerance

K+


An Ideal High Yielding Salinity Tolerant Variety

Highly tissue tolerant Good Excluder- Minimum per day uptake of Na+ High uptake of K+ per day Low Cl- uptake Low Na+/ K+ ratio Good initial vigour Agronomically superior with high yield potential (plant type

+ grain quality)


Breeding Strategy

Grouping of the genotypes based on the inherent physiological mechanism responsible for salinity tolerance

Inter-mating of the genotypes with high degree of expression of the contrasting salinity tolerance mechanism

Identifying / screening of the recombinants for pooling/ pyramiding of the mechanisms - MAS


RM283

R844

S2139RM23

RM140

RM113S1715S13994RM9

RM5C1456RM237RM246

0.0

27.428.4

40.0

64.9

66.2

71.2

75.3

77.291.998.299.1

103.1119.5123.5129.9

A

C52903S

C1733S

R2374B

C52903S

C1733S

R2374B

RM283

R844

S2139RM23

RM140

RM113S1715S13994RM9

RM5C1456RM237RM246

0.0

27.428.4

40.0

75.3

77.291.998.299.1

103.1119.5123.5129.9

RM283

R844

S2139RM23

RM140

RM113S1715S13994RM9

RM5C1456RM237RM246

0.0

27.428.4

40.0

75.3

77.291.998.299.1

103.1119.5123.5129.9

AP3206

RM3412CP03970

RM8094RM493CP6224

RM140

0.0

1.0

1.8

1.9

1.21.3

Short arm of chromosome 1

Progress of Saltot locus

• Saturated map of the Chromosome 1 (Saltol segment) is developed

• Closely linked markers linked to the saltol locus identified

• MAS is being validated in 3 breeding populations

60.6

(Source: Glenn B. Gregorio)


LOD threshold

RM140

CP6224

RM493

RM8094

CP03970

RM3412

CP010136

AP3206

0.0

b

a

Chromosome location of associated QTL of Salinity tolerance trait

2.5


• preprotein translocase, SecA subunit

• Sec23/Sec24 trunk domain, putative

• Ser Thr Kc

• Protein kinase domain

• S-adenosylmethionine synthetase

• chloroplast membrane protein

•Cold shock protein

• secretory peroxidase

• CBL-interacting protein kinase 19

• Peroxidase, putative

• Cell wall protein type (Extensin, Hydorxyproline rich, glycine rich)

• phospholipid/glycerol acyltransferase –like

• Mitochondrial carrier protein, putative

• GDSL-like Lipase/Acylhydrolase, putative

• organic cation transporter

• major facilitator superfamily protein

•Cell wall protein type (Extensin,Hydorxyproline rich, glycine rich)

• CP12 domain, putative

• Stress-inducible membrane pore protein

• Zinc finger, C3HC4 type (RING finger), putative

• Universal stress protein family

• Cation-chloride co-transporter

• Receptor like protein kinase

• Myb-like DNA-binding domain, putative

• Peroxidase, putative

• Cell wall protein type (Extensin,Hydorxyproline rich, glycine rich)

• Cation transporter

• Phospholipase D. Active site motif, putative

• Protein kinase domain, putative

• Dual specificity phosphatase, catalytic domain, putative

• Pectinemethyesterase/invertase inhibitor

• Pectinesterase

Rice Chromosome 1

60.6 60.9 62.5 64.9 65.4 66.2 67.6 67.9

cM

65.8

Saltol region ( Major QTL K+/Na+ratio )

(Source: Ellen Tumimbang)


11.9 Mb 12.13 Mb

12.11Mb 12.27Mb

12.25Mb 12.40Mb

12.0Mb 12.27 Mb

preprotein translocase, SecA subunit

Sec23/Sec24 trunk

WD40

Ser Thr Kc

Receptor like kinase

SAM synthetase

cold shock

protein

chloroplast

membrane protein

secretory peroxidase

CBL-interacting protein kinase 19

Peroxidase, putative

S_Tkc;WD40

0.27 Mb

SALtol Region ( Major QTL K+/Na+)

(~40 genes)

11.10Mb 12.7Mb

60.6 60.9 62.5 64.9 65.4 66.2 67.6 67.9

cM

65.8

Chromosome 1 of Rice

B1135C02

OSJNBa0011P19

P0426D06

B1153f04



List of genes that are located in the region of QTL and up-regulated by high salinity in rice

Gene nameInsertion

lines

Clone ID full length

cDNA

Rice 60k chip data under high salinity

(fold-induction)References

0.5 h 2 h 6 h

Pectinesterase 1B-23740,

1B-23741

CG408589

Ak105998 1.1 3.3 4.9

Ser/thr kinase AK065231 2.3 2.7 Guo et al., 2001

Phospholipase D 1515 AK120868 3.5 2.6 Kacperska, 2004

Zhu, 2002

SecA/protein transport factor

CL520490

CL520492

AK070488 3.1 1.5

Peroxidase AK099187 2.6 3.05 Pastori and Foyer, 2002

Sottosanto et al., 2004

Alkaline Invertase AK120720 4.0 2.2 4.2

Unknown cDNA AK099887 0.37 1.6 2.4(Source: Ellen Tumimbang)


• Putative SecA-type chloroplast protein transport factor

• Serine/threonine kinase

• Peroxidase

• Pectinesterase

• Phospholipase D. Active site motif -- putative

The position of the candidate genes in chromosome 1

60.6 60.9 62.5 64.9 65.4 66.2 67.6 67.9

cM

65.8

Saltol region ( Major QTL K+-Na+ratio )

Plant neutral/alkaline invertase



Mapping Salinity Tolerance Genes at Reproductive Stage

QTLs for salinity tolerance genes at seedling

stage are different from reproductive stage

• Seedling stage tolerance in chrom 1.

• Reproductive stage tolerance in chrom 3, 4, 7, and 9

Dr. Mirza M. Islam

Ph.D.


Salt tolerant rice varieties developed by IRRI and released in Philippines

IRRI 112 - PSBRc48 (Hagonoy) IRRI 113 - PSBRc50 (Bicol) IRRI 124 - PSBRc84 (Sipocot) IRRI 125 - PSBRc86 (Matnog) IRRI 126 - PSBRc88 (Naga) IRRI 128 - NSICRc106

Other salt-tolerant rice varieties

CSR10, CSR13, CSR23, CSR27, CSR30, CSR36 and Lunishree, Vytilla 1, Vytilla 2, Vytilla 3, Vytilla 4, Panvel 1, Panvel 2, Sumati, Usar dhan 1, 2 & 3 (India); BRRI dhan 40, BRRI dhan 41 (Bangladesh); OM2717, OM2517, OM3242 (Vietnam)


Realization of the Genetic Potential

Promote the Interdisciplinary IRRI-NARS collaborative research, based on CNRM technology and its validation in the farmers participatory mode

R

esp

onse

of

gen

otyp

es u

nd

er s

alt

stre

ss

envi

ron

men

t

Genetic Variation

True Potential of the salt tolerant rice genotype under salt-affected

soils

Seedling stage

tolerance

Vegetative stage

tolerance

Reproductive

stage tolerance

Cultural Practices

Nutrient Management with

respect to salt tolerant germplasm

Nu

rser

y / s

eed

lin

g M

anag

emen

t

Ad

ult

pla

nt

Man

agem

ent

Screen

ing from

available

genetic variab

ility

Molecu

lar Mark

ers

Physiological Mechanisms

Biochemical mechanisms Farmers’

opinion (PVS/PPB)


Progress in salinity research

= completed, = fast track, = not available /available /on-going

Saline Sodic Zn-def Acid

Donor Screening technique

Mechanism Genetics ? MAS development

Elite lines ?

Lab.

Field


Thanks for Your Kind Attention

Glenn B. Gregorio Rafiqul Islam Mirza M. Islam Jong C. KoR K Singh Andy Sajise Ghasem M. Nejad Glenn AlejarAdorada Dante Venus Elec Swe Thein MidieRhulyx Mendoza Jean Melgar Lorelie Ramos VenessaEllen Tumimbang Jaarmi Orly KelvinRollin De Ocampo Angelito Francisco


Please feel free to contact any time

[email protected]

R.K.Singh Plant Breeding Course, April 06 Breeding for Salt Tolerance in Rice R.K. Singh PBGB, IRRI.

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Transcript of R.K.Singh Plant Breeding Course, April 06 Breeding for Salt Tolerance in Rice R.K. Singh PBGB, IRRI.